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The emergence and spread of pathogens harboring extended spectrum beta-lactamase (ESBL) like carbapenem resistant Gram negative bacteria are the major emerging threat to public health. Of particular concern Klebsiella pneumoniae carbapenamase- producing strains have been recorded worldwide. Catheter associated urinary tract infections (CAUTI) caused by K. pneumoniae are significantly associated with morbidity and mortality. Hence the present work was aimed to develop a strategy for addressing these issues through an innovative approach of antibiofilm and immunomodulation. These two independent activities were analyzed in a Streptomyces derived ASK2 bioactive compound. While analysing the effect of sub-minimum inhibitory concentrations (sub-MICs), 0.5x of Minimum Inhibitory Concentration (MIC) was found to be more effective in preventing biofilm formation on coverslip and silicone catheter. The minimum biofilm eradication concentration (MBEC) was found to be 15-fold higher MIC with eradication of 75% of 3 day old biofilm. Apart from its antibiofilm potential, ASK2 also acts as an opsonin and enhances phagocytic response of macrophages against multidrug resistant K. pneumoniae. In addition, ASK2 resulted in elevated levels of nitric oxide generation by the macrophages and has a stimulating effect on IL-12, IFN-γ, and TNF-α proinflammatory cytokines. The opsonic role of ASK2 and its potential in modulating proinflammatory cytokines secreted by macrophages implies the importance of ASK2 in modulating cellular immune response of macrophages against MDR K. pneumoniae. The present study proposes ASK2 as a promising candidate for treating MDR K. pneumoniae infections with its dual properties of antibiofilm and immunomodulatory activities.

The emergence and spread of multi-drug resistant (MDR) especially carbapenem-resistant Klebsiella pneumoniae is a major emerging threat to public health, leading to excess in mortality rate as high as 50–86%. MDR K. pneumoniae manifests all broad mechanisms of drug resistance, hence development of new drugs to treat MDR K. pneumoniae infection has become a more relevant question in the scientific community. In the present study a potential Streptomyces sp. ASK2 was isolated from rhizosphere soil of medicinal plant. The multistep HPLC purification identified the active principle exhibiting antagonistic activity against MDR K. pneumoniae. The purified compound was found to be an aromatic compound with aliphatic side chain molecule having a molecular weight of 444.43 Da. FT-IR showed the presence of OH and C=O as functional groups. The bioactive compound was further evaluated for drug induced toxicity and efficacy in adult zebrafish infection model. As this is the first study on K. pneumoniae – zebrafish model, the infectious doses to manifest sub-clinical and clinical infection were optimized. Furthermore, the virulence of K. pneumoniae in planktonic and biofilm state was studied in zebrafish. The MTT assay of ex vivo culture of zebrafish liver reveals non-toxic nature of the proposed ASK2 compound at an effective dose. Moreover, significant increase in survival rate of infected zebrafish suggests that ASK2 compound from a new strain of Streptomyces sp. was potent in mitigating MDR K. pneumoniae infection.

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44Infectious bacteria in biofilm mode are involved in many of persistent infections. 45 Owing to its importance in clinical settings many in vitro and in vivo studies have analysed 46 the structural and functional properties of biofilm, its resistance to antibiotic exposure etc. 47 Currently the immune mechanism toward the clearance of biofilm infections is being 48 investigated. K. pneumoniae is one of the major leading causes of biofilm infections on 49 indwelling medical devices. There was no previous literature that demonstrates the 50 interactions of macrophage cells lines and Klebsiella biofilm, as the first report, we 51 investigated the in vitro response of Klebsiella biofilm to phagocytosis and cytokine 52 expression. We developed an in vitro model to study the interactions of Kebsiella biofilm 53 and macrophage. The phagocytosis assay was performed for heat inactivated and live 54 biofilm. A similar phagocytic response against both biofilms were observed when these 55 cells were exposed to RAW 264.7 macrophages. Also, the expressions of TLR2, iNOS, 56 inflammatory cytokines such as IL-β1, IFN-γ, IL-6, IL-12, IL-4, TNF-α and anti-57 inflammatory cytokines, IL-10 during phagocytosis were analysed. These results 58 collectively demonstrated that the rate of phagocytosis was an average of 15% for both 59 biofilms. Also, when activated macrophage was exposed to heat-inactivated or live biofilms, 60 there was a significant increase in proinflammatory cytokine genes together with expected 61 increase in TLR2 and iNOS. Thus, it is clear that macrophage response against biofilm 62 producing K. pneumoniae results in increase in phagocytic rate and a corresponding increase 63 in inflammatory cytokine gene expression which could be important for clearing K. 64 pneumoniae cells.65 Introduction 68 K. pneumoniae is a Gram-negative, encapsulated opportunistic pathogen that 69 colonizes almost every part of the human body with most preferred site being the respiratory, 70 gastrointestinal and urinary tracts [1]. K. pneumoniae causes both hospital and community-71 acquired infections [2]. Pneumonia, meningitis, urinary tract infections and catheter-related 72 bloodstream infections are the potential illness caused by this bacterium [3]. The major risk 73 factors associated with K. pneumoniae infection includes central venous catheterization, 74 urinary catheterization, mechanical ventilation, prolonged stay in intensive-care unit, low 75 birth weight in preterm infants and individuals with impaired immunity [4]. 76 Klebsiella spp are characterized by the presence of capsular polysaccharides (CPS), 77 type 1 and 3 fimbriae as the major virulence factors. These cellular components play an 78 important role in the adhesion and colonization of host tissues. In addition, these virulence 79 factors are essential for biofilm formation on indwelling medical devices and persistent 80 infections [2]. 81 In order to overcome the infections caused by planktonic and biofilm of K. 82 pneumoniae, both humoral and cell-mediated...